This project is designed to develop a novel mouse model to study mitochondrial dynamics and function. Genetic engineering and molecular biological techniques will be utilized to create "transmitochondrial" mice. Procedures for mitochondrial transfer will lead to a greater understanding of mitochondrial dynamics, leading to the development of novel strategies and therapies for human metabolic diseases influenced by aberrations in mitochondrial function or mutation. To make a mitochondrial disease model, the ability to manipulate normal and mutant mitochondria in vivo is a critical first step. While gene transfer has been performed in a host of cell types and organisms, transfer of nuclear DNA has been the only demonstrable form of mammalian gene transfer to date. With the advent of gene transfer technologies and PCR analyses, this project targets the development of technology to establish mitochondria transfer as a viable technique to genetically engineer mouse models. Initially, these models will serve to explore mitochondrial dynamics in an in vivo system. Ultimately, transmitochondrial mice will be used to explore the role of the mitochondrial genome in human metabolic disease processes and potentially in the development of novel human gene therapies. Here, we propose to establish a means of mitochondrial transfer and to produce mice harboring a foreign mitochondrial genome. Our specific aims include: (1) the isolation, purification and quantification of mitochondria from two strains of mice possessing mitochondrial genomes with discernable differences in nucleotide sequences and restriction enzyme digestion patterns (Mus domesticus and Mus spretus), (2) the establishment of a fibroblast cell line for efficient production of specific mitochondria to augment gene transfer procedures, (3) the development of mitochondria transfer procedures using the mouse model, and (4) initial characterization of transmitochondrial mouse lines created during the course of this project.